The aim of this paper was to gain further insight into the effect of the clay pretreatment process oil photofermentative hydrogen production. This two-stage process involved a clay pretreatment step followed by photofermentation. which was performed under anaerobic conditions with the illumination by Tungsten lamps. Rhodobacter sphaeroUts O.U.001 was used for photofermentation. Higher amounts of color (65%), total phenol (81%) and chemical oxygen demand (3%) removal efficiencies were achieved after clay pretreatment process. During photofermentative hydrogen production with the effluent of clay pretreatment process, the main organic compounds resulting higher hydrogen production rates were found to be acetic, lactic, propionic, and butyric acids. Compared to photofermentation using raw olive mill wastewater (16 L-H2/L-OMW), the amount of photofermentative hydrogen production was doubled by using the effluent of the clay pretreatment process (31.5 L-H2/L-OMW). The reasons for the improvement of hydrogen production by clay treatment call be attributed to the high removal of the hardly biodegradable compounds such as phenols; minor removal of organic acids, sugars and amino acids that are known to enhance photofermentative hydrogen production; and the color depletion of raw OMW which might cause a shadowing effect on the photosynthetic bacteria. (C) 2008 Elsevier Ltd. All rights reserved.